Anelloviruses are small, single-stranded, circular DNA viruses that infect a wide range of animal species from humans to domestic animals including pigs (Hino, S., and H. Miyata. 2007. Torque teno virus (TTV): current status. Rev Med Virol 17:45-57; Okamoto, H. 2009. TT viruses in animals. Curr Top Microbiol Immunol 331:35-52). Most recently, all human and other animal anelloviruses have been assigned into a newly established family Anelloviridae that includes nine genera (Biagini, P., M. Bendinelli, S. Hino, L. Kakkola, A. Mankertz, C. Niel, H. Okamoto, S. Raidal, C. G. Teo, and D. Todd. 2011. Anelloviridae, p. 331-341. In A. M. Q. King, M. J. Adams, E. B. Carstens, and E. J. Lefkowitz (ed.), Virus Taxonomy, 9th Report of the ICTV. Elsevier Academic Press, London). Human anelloviruses include Torque teno virus (TTV), Torque teno mini virus (TTMV) and Torque teno midi virus (TTMDV) that belong to three different genera. Human TTV, TTMV and TTMDV are non-enveloped spherical viruses with DNA genomes of 3.6-3.9, 2.8-2.9 and 3.2 kb in length, respectively (Okamoto, H. 2009. History of discoveries and pathogenicity of TT viruses. Curr Top Microbiol Immunol 331:1-20). These three groups of human anelloviruses show a high degree of genetic diversity, and infections of TTV, TTMV and TTMDV at a high prevalence in human populations have been documented worldwide (Ninomiya, M., M. Takahashi, T. Nishizawa, T. Shimosegawa, and H. Okamoto. 2008. Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy. J Clin Microbiol 46:507-14; Okamoto, H. 2009. History of discoveries and pathogenicity of TT viruses. Curr Top Microbiol Immunol 331:1-20). On the other hand, porcine anelloviruses or Torque teno sus viruses (TTSuV) is assigned into a new genus Iotatorquevirus comprising two species (TTSuV1 and TTSuV2), each also characterized by high genetic diversity with a genomic size of approximately 2.8 kb (Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV. Virology 396:287-97, Niel, C., L. Diniz-Mendes, and S. Devalle. 2005. Rolling-circle amplification of Torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup. J Gen Virol 86:1343-7). TTSuV1 and TTSuV2 are highly prevalent in pig populations in many countries (Gallei, A., S. Pesch, W. S. Esking, C. Keller, and V. F. Ohlinger. 2010. Porcine Torque teno virus: determination of viral genomic loads by genogroup-specific multiplex rt-PCR, detection of frequent multiple infections with genogroups 1 or 2, and establishment of viral full-length sequences. Vet Microbiol 143:202-12; Kekarainen, T., M. Sibila, and J. Segales. 2006. Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain. J Gen Virol 87:833-7; McKeown, N. E., M. Fenaux, P. G. Halbur, and X. J. Meng. 2004. Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries. Vet Microbiol 104:113-7).
Human and porcine anelloviruses share the same genomic structure, which consists of at least four presumed open reading frames (ORFs), ORF1, ORF2, ORF1/1 and ORF2/2, as well as a short stretch of high GC content in the untranslated region (UTR) (Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV. Virology 396:287-97; Okamoto, H., M. Takahashi, T. Nishizawa, A. Tawara, K. Fukai, U. Muramatsu, Y. I Saito, and A. Yoshikawa. 2002. Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias. J Gen Virol 83:1291-7; 39. Qiu, J., L. Kakkola, F. Cheng, C. Ye, M. Soderlund-Venermo, K. Hedman, and D. J. Pintel. 2005. Human circovirus TT virus genotype 6 expresses six proteins following transfection of a full-length clone, J Virol 79:6505-10). The transcription pattern and related translational products of human TTV genogroup 1 have been experimentally determined by using two full-length TTV DNA clones (Mueller, B., A. Maerz, K. Doberstein, T. Finsterbusch, and A. Mankertz. 2008. Gene expression of the human Torque Teno Virus isolate P/1C1. Virology 381:36-45; 39. Qiu, J., L. Kakkola, F. Cheng, C. Ye, M. Soderlund-Venermo, K. Hedman, and D. J. Pintel. 2005. Human circovirus TT virus genotype 6 expresses six proteins following transfection of a full-length clone, J Virol 79:6505-10). It was shown that the human TTV genome expresses three or more spliced mRNAs encoding at least six proteins, ORF1, ORF2, ORF1/1, ORF2/2, ORF1/2 and ORF2/3 (Mueller, B., A. Maerz, K. Doberstein, T. Finsterbusch, and A. Mankertz. 2008. Gene expression of the human Torque Teno Virus isolate P/1C1. Virology 381:36-45). The transcriptional analysis and protein expression profile using cloned full-length genomic DNA have not been reported for TTSuV.
The ORF1 of TTSuV is believed to encode a viral capsid and replication-associated protein (Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV. Virology 396:287-97; Okamoto, H., M. Takahashi, T. Nishizawa, A. Tawara, K. Fukai, U. Muramatsu, Y. I Saito, and A. Yoshikawa. 2002. Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias. J Gen Virol 83:1291-7). IgG antibodies against the ORF1 of TTV and TTSuV have been detected in human and pig sera, respectively (15. Huang, Y. W., K. K. Harrall, B. A. Dryman, N. M. Beach, S. P. Kenney, T. Opriessnig, E. M. Vaughn, M. B. Roof, and X. J. Meng. 2011. Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral load and IgG antibody level in pigs. Virus Res 158:79-88; Kakkola, L., H. Boden, L. Hedman, N. Kivi, S. Moisala, J. Julin, J. Yla-Liedenpohja, S. Miettinen, K. Kantola, K. Hedman, and M. Soderlund-Venermo. 2008. Expression of all six human Torque teno virus (TTV) proteins in bacteria and in insect cells, and analysis of their IgG responses. Virology 382:182-9; 38. Ott, C., L. Duret, I. Chemin, C. Trepo, B. Mandrand, and F. Komurian-Pradel. 2000. Use of a TT virus ORF1 recombinant protein to detect anti-TT virus antibodies in human sera. J Gen Virol 81:2949-58).
The pathogenic potential of anellovirus is still controversial. Currently, human TTV is not considered to be directly associated with a particular disease, although recent studies suggested TTV may serve as an immunological trigger of multiple sclerosis (Maggi, F., and M. Bendinelli. 2010. Human anelloviruses and the central nervous system. Rev Med Virol 20:392-407). Similarly, whether TTSuV is associated with a swine disease is still debatable. TTSuV1 was shown to partially contribute to the experimental induction of porcine dermatitis and nephropathy syndrome (PDNS) and postweaning multisystemic wasting syndrome (PMWS or porcine circovirus associated disease, PCVAD) in a gnotobiotic pig model (Ellis, J. A., G. Allan, and S. Krakowka. 2008. Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs. Am J Vet Res 69:1608-14; 22. Krakowka, S., C. Hartunian, A. Hamberg, D. Shoup, M. Rings, Y. Zhang, G. Allan, and J. A. Ellis. 2008. Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2. Am J Vet Res 69:1615-22). PMWS-affected pigs in Spain had a higher prevalence and viral loads of TTSuV2 than the PMWS-unaffected pigs (Aramouni, M., J. Segales, M. Sibila, G. E. Martin-Valls, D. Nieto, and T. Kekarainen. 2011. Torque teno sus virus 1 and 2 viral loads in postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) affected pigs. Vet Microbiol 153:377-81; 21. Kekarainen, T., M. Sibila, and J. Segales. 2006. Prevalence of swine Torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain. J Gen Virol 87:833-7). Moreover, a significantly lower level of anti-TTSuV2 antibody was found in PCVAD-affected pigs than in PCVAD-unaffected pigs (Huang, Y. W., K. K. Harrall, B. A. Dryman, N. M. Beach, S. P. Kenney, T. Opriessnig, E. M. Vaughn, M. B. Roof, and X. J. Meng. 2011. Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral load and IgG antibody level in pigs. Virus Res 158:79-88). However, results from other studies did not support a direct association of TTSuV1 or TTSuV2 with PCVAD or association of type 2 porcine circovirus (PCV2) and TTSuV with porcine reproductive failures (Gauger, P. C., K. M. Lager, A. L. Vincent, T. Opriessnig, M. E. Kehrli, Jr., and A. K. Cheung. 2011. Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b. Vet Microbiol 153:229-39; Huang, Y. W., K. K. Harrall, B. A. Dryman, T. Opriessnig, E. M. Vaugh, M. B. Roof, and X. J. Meng. 2012. Serological profile of Torque teno sus virus species 1 (TTSuV1) in pigs and antigenic relationships between two TTSuV1 genotypes (1a and 1b), between two species (TTSuV1 and 2), and between porcine and human anelloviruses. J. Virol. Submitted Manuscript; Lee, S. S., S. Sunyoung, H. Jung, J. Shin, and Y. S. Lyoo. 2010. Quantitative detection of porcine Torque teno virus in Porcine circovirus-2-negative and Porcine circovirus-associated disease-affected pigs. J Vet Diagn Invest 22:261-4; Ritterbusch, G. A., C. A. Sa Rocha, N. Mores, N. L. Simon, E. L. Zanella, A. Coldebella, and J. R. Ciacci-Zanella. 2011. Natural co-infection of torque teno virus and porcine circovirus 2 in the reproductive apparatus of swine. Res Vet Sci. doi:10.1016/j.rvsc.2011.04.001).
Due to the lack of a cell culture system to propagate anelloviruses, little is known regarding the molecular biology and pathogenesis of anelloviruses. In order to definitively characterize diseases associated with anellovirus infection, an appropriate animal model is needed. Since multiple infections of different genotypes or subtypes of human TTV or TTSuV are common events (Gallei, A., S. Pesch, W. S. Esking, C. Keller, and V. F. Ohlinger. 2010. Porcine Torque teno virus: determination of viral genomic loads by genogroup-specific multiplex rt-PCR, detection of frequent multiple infections with genogroups 1 or 2, and establishment of viral full-length sequences. Vet Microbiol 143:202-12; Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV. Virology 396:287-97; Ninomiya, M., M. Takahashi, T. Nishizawa, T. Shimosegawa, and H. Okamoto. 2008. Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy. J Clin Microbiol 46:507-14), a biologically pure and isolated form of a specific anellovirus generated from full-length infectious DNA clone is also required for a pathological study of a single phenotype. Although infectious DNA clones of human TTV in cultured cells have been reported (de Villiers, E. M., S. S. Borkosky, R. Kimmel, K. Gunst, and J. W. Fei. 2011. The diversity of torque teno viruses: in vitro replication leads to the formation of additional replication-competent subviral molecules. J Virol 85:7284-95; Kakkola, L., J. Tommiska, L. C. Boele, S. Miettinen, T. Blom, T. Kekarainen, J. Qiu, D. Pintel, R. C. Hoeben, K. Hedman, and M. Soderlund-Venermo. 2007. Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTN) genotype6. FEBS J 274:4719-30; Leppik, L., K. Gunst, M. Lehtinen, J. Dillner, K. Streker, and E. M. de Villiers. 2007. In vivo and in vitro intragenomic rearrangement of TT viruses. J Virol 81:9346-56), it is important to construct an infectious TTSuV DNA clone so that TTSuV can be used as a useful model to study the replication and transcription mechanisms and to dissect the structural and functional relationships of anellovirus genes. More importantly, the availability of a TTSuV infectious DNA clone will afford us an opportunity to use the pig as a model system to study the replication and pathogenesis of TTSuV or even human TTV.
Multiple infections of human TTV with different genotypes in a single human individual or TTSuV with different genotypes or subtypes in a single pig have been documented (Ball, J. K., R. Curran, S. Berridge, A. M. Grabowska, C. L. Jameson, B. J. Thomson, W. L. Irving, and P. M. Sharp. 1999. TT virus sequence heterogeneity in vivo: evidence for co-infection with multiple genetic types. J Gen Virol 80 (Pt 7): 1759 68; Forms, X., P. Hegerich, A. Darnell, S. U. Emerson, R. H. Purcell, and J. Bukh. 1999. High prevalence of TT virus (TTV) infection in patients on maintenance hemodialysis: frequent mixed infections with different genotypes and lack of evidence of associated liver disease. J Med Virol 59:313-7; Gallei, A., S. Pesch, W. S. Esking, C. Keller, and V. F. Ohlinger. 2010. Porcine Torque teno virus: determination of viral genomic loads by genogroup-specific multiplex rt-PCR, detection of frequent multiple infections with genogroups 1 or 2, and establishment of viral full-length sequences. Vet Microbiol 143:202-12; Huang, Y. W., Y. Y. Ni, B. A. Dryman, and X. J. Meng. 2010. Multiple infection of porcine Torque teno virus in a single pig and characterization of the full-length genomic sequences of four U.S. prototype PTTV strains: implication for genotyping of PTTV. Virology 396:289-97; Jelcic, I., A. Hotz-Wagenblatt, A. Hunziker, H. Zur Hausen, and E. M. de Villiers. 2004. Isolation of multiple TT virus genotypes from spleen biopsy tissue from a Hodgkin's disease patient: genome reorganization and diversity in the hypervariable region. J Virol 78:7498-507; Niel, C., F. L. Saback, and E. Lampe. 2000. Coinfection with multiple TT virus strains belonging to different genotypes is a common event in healthy Brazilian adults. J Clin Microbiol 38:1926-30; Ninomiya, M., M. Takahashi, T. Nishizawa, T. Shimosegawa, and H. Okamoto. 2008. Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy. J Clin Microbiol 46:507-14). These findings raise the question whether the anti-ORF1 capsid antibodies recognized by the antigen from a particular TTV or TTSuV species/geno types also comprise anti-ORF1 antibodies against other distinct TTV or TTSuV species/genotypes and whether the anti-ORF1 antibodies from one TTV or TTSuV genotype can cross-protect against infection with another genotype. To our knowledge, for human TTV or TTSuV infection there is no information on this topic available to date. Furthermore, the antigenic diversity and relationship of anelloviruses have never been assessed (Maggi, F., and M. Bendinelli. 2009. Immunobiology of the Torque teno viruses and other anelloviruses. Curr Top Microbiol Immunol 331:65-90). It is reasonable to speculate that there is little, if any, antigenic cross-reactivity between different anellovirus species/genotypes, due to the fact that concurrent infections with multiple anelloviruses in a single individual or animal exist.
The inventors have previously developed and validated serum Western blot (WB) and indirect ELISA assays for detection of the IgG antibody against TTSuV2 in porcine sera using the purified recombinant TTSuV2-ORF1 protein expressed in E. coli (Huang, Y. W., K. K. Harrall, B. A. Dryman, N. M. Beach, S. P. Kenney, T. Opriessnig, E. M. Vaughn, M. B. Roof, and X. J. Meng. 2011. Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral load and IgG antibody level in pigs. Virus Res 158:79-88). By using TTSuV2-specific real-time quantitative PCR (qPCR) and ELISA, The inventors further presented the combined virological and serological profile of TTSuV2 infection under natural or diseased conditions using 160 porcine sera collected from different sources (Huang, Y. W., K. K. Harrall, B. A. Dryman, N. M. Beach, S. P. Kenney, T. Opriessnig, E. M. Vaughn, M. B. Roof, and X. J. Meng. 2011. Expression of the putative ORF1 capsid protein of Torque teno sus virus 2 (TTSuV2) and development of Western blot and ELISA serodiagnostic assays: correlation between TTSuV2 viral load and IgG antibody level in pigs. Virus Res 158:79-88). In the present invention, The inventors initially aimed to assess the serological profiles of the two TTSuV1 genotypes (TTSuV1a and TTSuV1b) in pigs, respectively. Subsequently, the inventors aimed to compare the virological and serological profiles of TTSuV1a and TTSuV1b with that of TTSuV2, and to determine the degree of correlation of IgG antibody levels between anti-TTSuV1a and -TTSuV1b and between anti-TTSuV1a or -1b and anti-TTSuV2. Finally, for the first time, the inventors assessed the antigenic relationships between two TTSuV1 genotypes (TTSuV1a and TTSuV1b), between two species (TTSuV1 and TTSuV2), and between porcine and human genogroup 1 anelloviruses using ELISA and immunofluorescence assay with antibody cross-reactions in PK-15 cells transfected with recombinant plasmids expressing the ORF1s from TTSuV1a, TTSuV1b and TTSuV2, respectively.